Sirtuins are crucial regulators of T cell metabolism and functions

It is well known that metabolism underlies T cell differentiation and functions. The pathways regulating T cell metabolism and function are interconnected, and changes in T cell metabolic activity directly impact the effector functions and fate of T cells. Thus, understanding how metabolic pathways influence immune responses and ultimately affect disease progression is paramount. Epigenetic and posttranslational modification mechanisms have been found to control immune responses and metabolic reprogramming. Sirtuins are NAD+-dependent histone deacetylases that play key roles during cellular responses to a variety of stresses and have recently been reported to have potential roles in immune responses. Therefore, sirtuins are of significant interest as therapeutic targets to treat immune-related diseases and enhance antitumor immunity. This review aims to illustrate the potential roles of sirtuins in different subtypes of T cells during the adaptive immune response.

INTRODUCTION
Overview of the T cell-mediated adaptive immune response The adaptive immune response elicited by T cells is crucial in mounting specific immune responses against foreign pathogens. T cells develop in the thymus and, upon maturation, are classified by their expression of either CD4 or CD8 receptors. CD4 + and CD8 + T cells exist in several subsets that perform unique functions during an immune response and are characterized by specific surface receptors, cytokine secretion, and expression of lineagedefining transcription factors ( Fig. 1) 1,2 . Depending on the pathogen type and the cytokines generated by antigenpresenting cells (APCs), CD4 + T cells can become activated and differentiate into functionally distinct T helper (Th) 1, Th2, Th17, Th9, or T regulatory (Treg) cells 3 , while CD8 + T cells differentiate into cytotoxic T lymphocytes (CTLs) 4 . Th1 cells, which produce interferon-gamma (IFN-γ), interleukin (IL)-2, and tumor necrosis factor-beta (TNF-β), evoke cell-mediated immunity and phagocyte-dependent inflammation and are involved in the elimination of intracellular pathogens 5 . Th2 cells, which produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, are known to provide immunity against extracellular parasites, including helminths, and play major roles in the induction and persistence of asthma as well as other allergic diseases 6 . Th9 cells are known to primarily produce IL-9 and facilitate the immune response against helminth parasites 7 . Th17 cells are characterized by the production of IL-17 and are involved in host protection against microbial infections that are not resolved by Th1 or Th2 immunity, including infections with a subset of extracellular bacteria and some fungi 8 . Retinoic acid receptor-related orphan nuclear receptor gamma (RORγt) is considered to be one of the master regulators of the development of Th17 cells 8 . In contrast to the proinflammatory phenotype of Th cells, Treg cells participate in the prevention of uncontrollable inflammatory responses, autoimmune diseases and allergies by suppressing T effector (T EFF ) cells and other immune cells 9 . Treg cells are defined by the expression of the forkhead box P3 (Foxp3) transcription factor 10 . This cell subset secretes key antiinflammatory cytokines, including IL-10, tumor growth factor-β (TGF-β), and IL-35. CTLs are CD8 + T EFF cells that participate in cellular immunity, and their role is to directly kill infected or malignant cells through the release of cytotoxic cytokines, including TNF-α and IFN-γ, as well as granules including perforin and granzymes 4 .

Metabolic shifts in T cells
Naive T (T N ) cells are quiescent and require energy only for survival and circulation. These cells rely predominantly on mitochondrial oxidative phosphorylation (OxPhos) metabolism to generate ATP from glucose or fatty acids (FAs) 11 . Activation of T cells occurs after stimulation of the T cell antigen receptor (TCR) by a ligand in association with costimulatory signals, which initiates several signaling pathways that promote cell differentiation and growth. This activation is associated with a transition from relatively quiescent oxidative metabolism to intense glycolytic metabolism to support energetic needs for proliferation and cytokine production [11][12][13] . In addition to increased glucose catabolism in activated T cells, glutamine uptake and glutaminolysis are also upregulated to supply the tricarboxylic acid (TCA) cycle for ATP generation 14,15 . The metabolic profile of each specialized T cell subset is optimized to support their unique functions. T EFF cells, including Th1, Th2, and Th17 cells and CTLs, principally rely on aerobic glycolysis and glutaminolysis to promote their rapid growth, proliferation, and effector functions 16,17 . In contrast, Treg cells rely primarily on FA oxidation (FAO) and glutaminolysis to support their suppressive activity 18,19 .
Following the primary immune response, a portion of CD4 + and CD8 + T cells become memory T (T M ) cells that remain ready to rapidly respond to the same antigen. These cells are long-lived, and until reactivation, they exhibit relatively quiescent oxidative metabolism fueled by FAO, similar to that of T N cells ( Fig. 1) 20,21 . Due to competition for glucose and glutamine with cancer cells, tumor-infiltrating T lymphocytes (TILs) are metabolically compromised and functionally exhausted within the tumor microenvironment (TME) 22 . To partially rescue their effector functions, TILs have been shown to increase FAO metabolism as a way to utilize alternative fuel sources 23 .
Increasing evidence indicates that immune cell identity, functions, and metabolism are mediated by overlapping signaling pathways that are controlled, at least in part, via epigenetic and posttranslational modification (PTM) mechanisms. Sirtuins are key epigenetic and PTM regulators in T cells. In this review, we will provide relevant insights into the current understanding of T cellspecific immune response regulation by sirtuins and the therapeutic potential of sirtuin modulators in immune-related diseases.

EPIGENETIC MECHANISMS
In multicellular organisms, all cells contain the same genome; however, gene expression profiles vary from cell to cell, which contribute to differentiation into various cell types within the same organism 24 . This cell type differentiation is mostly regulated by epigenetic mechanisms, which result in dynamic changes in gene expression patterns without changing the DNA sequence 25 . Epigenetic regulation of gene expression is known to occur at the DNA, histone, and RNA levels. In this context, DNA methylation, histone methylation, acetylation, ubiquitination, phosphorylation, and microRNA-dependent gene silencing have been well characterized 26 . Methylation of DNA is mediated by DNA methyltransferases and consists of the addition of a methyl group to cysteine residues within DNA regions that are rich in cysteineguanine dinucleotides. DNA methylation can act either to inhibit gene transcription if methylation occurs within a promoter region or to promote gene transcription if methylation occurs within the gene body 27 . Noncoding microRNAs are single-stranded RNA fragments that interact with target messenger (m)RNAs via a perfectly complementary base sequence, resulting in mRNA cleavage and translational repression 28 . Another important and common epigenetic mechanism that contributes to gene expression regulation is the modification of histones. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are the two classes of enzymes involved in the dynamic regulation of histone acetylation 29 . Acetylated histones maintain an open and fluid chromatin structure, called euchromatin, promoting gene transcription, while deacetylated histones often form a tightly packed chromatin structure that prevents gene transcription, called heterochromatin 30 .

POSTTRANSLATIONAL MODIFICATION
PTM is a biochemical mechanism in which amino acid residues from a protein are covalently modified after translation to regulate protein folding, degradation, signaling, localization, stability, enzymatic activity or protein-protein interactions 31 . There are over 400 known PTM types, including phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis, which influence almost all aspects of physiological and pathological cell processes 32 . In contrast to epigenetic mechanisms, PTM allows rapid changes in protein properties in response to cellular needs during acute stress phases. In addition, multisite PTM leads to a vast variety of potential molecular states 33 . Phosphorylation is the most widely studied PTM and can regulate signaling pathways, the cell cycle, metabolism, the immune response, and cellular growth and differentiation 34 . Ubiquitin (Ub) is a small and highly conserved protein made of 76 amino acids that can be attached to a lysine substrate through a complex conjugation process requiring a Ubactivating enzyme (E1), Ub-conjugating enzyme (E2) and Ub ligase (E3). Ubiquitination plays important regulatory roles in the life cycle of proteins by leading to the degradation of target substrates 35 . This process can be reversed by deubiquitinating enzymes (DUBs), which remove conjugated Ub molecules from target substrates 36 . Protein acetylation has emerged as a key PTM participating in cellular regulation, particularly through the modification of histones, nuclear transcription regulators and metabolic enzymes 37 . Lysine acetylation is the prevalent modification of metabolic enzymes, and virtually every enzyme in glycolysis, gluconeogenesis, the TCA cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism has been found to be acetylated in human liver tissue 38 . Protein acetylation status is regulated by a highly dynamic equilibrium between HATs and HDACs 39 .

OVERVIEW ON SIRTUINS
The sirtuin proteins are classified within class III HDACs, which require nicotinamide adenine dinucleotide (NAD + ) as a cofactor for their deacetylase activity. Although initially identified as HDACs, later studies revealed that sirtuins can deacetylate a variety of nonhistone proteins 40 . Moreover, sirtuins show additional enzymatic functions other than deacetylation.
In mammals, seven sirtuins (Sirt1-7) are ubiquitously expressed with distinct subcellular localization and functions (Fig. 2) [41][42][43] . Sirt1 is the most studied mammalian sirtuin and is primarily localized in the nucleus. However, under specific conditions, Sirt1 can be transported to the cytoplasm 44 . Sirt2 is a predominantly cytosolic sirtuin that can migrate to the nucleus during mitosis 45 . Sirt6 is exclusively localized in the nucleus, whereas Sirt7 is localized in the nucleolus 41 . All the remaining sirtuins (3)(4)(5) are localized in mitochondria 46 . All sirtuins except Sirt5 have mono-ADP-ribosyl transferase activity, whereas Sirt5 catalyzes the Fig. 1 The metabolic programs of CD4 + T cell subsets. Distinct T cell subsets utilize specific metabolic programs to support their functions. Each functional subset is characterized by specific signaling pathways, transcription factors, metabolic programs, and effector cytokines. Naive T cells are quiescent and rely on OxPhos for their minimal energetic needs. Upon activation, activated T cells switch to aerobic glycolysis and increase glucose and glutamine uptake, which supports cell growth and proliferation. The differentiation of activated T cells into different subsets is due to several metabolic and signaling pathways. Th1, Th2, Th9, and Th17 cells primarily rely on aerobic glycolysis and glutaminolysis; in contrast, Treg and T M cells upregulate fatty acid oxidation.
The first sirtuin protein was discovered in yeast as silent information regulator 2 (Sir2), whose homolog in mammals is known as Sirt1 49 . Since it was discovered that Sir2 extends yeast lifespan in response to calorie restriction, sirtuins have received considerable attention as anti-aging regulators 50 . Furthermore, sirtuins have been described to regulate a variety of cell processes, including genome stability and cell metabolism 42,43 , and are currently gaining extensive interest for their role in mediating several diseases associated with inflammation 51 , metabolic disorders 52 , and cancers 53 (Fig. 2).
As sirtuins depend on NAD + (a critical cofactor of metabolism) for their enzymatic activity, any fluctuations in NAD + levels related to nutrient availability affect their functions 54 . Hence, sirtuins serve as energy sensors that directly link environmental signals to cellular metabolic homeostasis. Given the link between metabolic reprogramming, nutrient availability and T cell effector function 55 , sirtuins are speculated to be potential regulators of the immune response, impacting the ability of the immune system to combat foreign pathogens or malignant cells. While each of the sirtuins has been broadly studied and demonstrated to be involved in a number of diseases associated with inflammation 56 , investigations of the roles of sirtuins in adaptive immunity have been limited to certain aspects of the immune response, and the most intense attention has been given to Sirt1 and Sirt3 57 . This review will discuss the latest advances in the roles of the seven mammalian sirtuins in the T cell adaptive response and include our considerations for targeting sirtuins to manipulate the immune response.

SIRT1
Most studies on Sirt1 have indicated that decreased Sirt1 expression or activity contributes to the enhancement of T cell activation, thereby leading to the occurrence of autoimmune diseases 58 . The immunoregulatory function of Sirt1 is dependent on the cell types and specific substrates that are targeted within the immune response. In addition, Sirt1-mediated regulation of metabolic processes is critical for optimal immune cell function 59 .
c-Jun is upregulated after T cell activation to induce IL-2 production, cell proliferation and differentiation 60 . An early study indicated that Sirt1 inhibits the immune response by blocking c-Jun transcription factor activity, which supports IL-2 production 61 and thereby decreases Th1 cell activation. Indeed, enhanced expression and activity of Sirt1 induced by resveratrol treatment impeded CD4 + T cell activation and IFN-γ production, further confirming that Sirt1 negatively impacts Th1 differentiation and IFN-γ secretion 62 . More recently, in a mouse model of ovarian cancer, Th1 cell differentiation of CD4 + T cells was found to be increased after treatment with artesunate, a promoter of microRNA-142 expression that downregulates Sirt1 expression, again confirming the suppressive role of Sirt1 in Th1 cell differentiation 63 .
Studies in Sirt1 knockout (Sirt1 −/− ) mice showed that Sirt1 −/− T cells display increased proliferation and IL-2 production, and Sirt1 −/− mice are more susceptible to developing autoimmune diseases 64 . Further studies indicated that IL-2 can reverse T cell anergy by suppressing Sirt1 transcription via cytosolic sequestration of its upstream transcription factor, FoxO3a. The expression of a constitutively active form of FoxO3a blocks IL-2-mediated reversal of T cell tolerance by retaining Sirt1 expression 65 .
B-cell lymphoma 2-associated factor 1 (Bclaf1), primarily considered a promoter of cellular apoptosis, has been found to be critical for T cell activation 66 . Kong et al. reported that Sirt1 suppresses Bclaf1 activity by deacetylating histone lysine residues at the Bclaf1 promoter region, resulting in decreased IL-2 gene transcription. Accordingly, Sirt1 −/− T cells displayed higher expression of the Bclaf1 gene and IL-2 production, and specific knockdown of Bclaf1 reversed the increase in IL-2 production and proliferation observed in Sirt1 −/− T cells 67 .
Early studies using ovalbumin-induced asthma models in mice demonstrated that pharmacologic inhibition of Sirt1 reduced allergic reactions compared with mock treatment 68,69 . Furthermore, Sirt1 inhibition was found to suppress the differentiation of Th2 cells through the B cell lymphoma/leukemia 11B (Bcl11b) transcriptional activator. Bcl11b is essential for Th2 differentiation, and mice lacking Bcl11b in mature T cells have a diminished capacity to mount Th2 responses during helminth infection and allergic asthma 70 . Sirt1 interacts directly with Bcl11b and is recruited to the promoter template in a Bcl11b-dependent manner to deacetylate histones, leading to transcriptional repression 71 .
Hypoxia-inducible factor 1-alpha (HIF-1α) activity has consistently been associated with the generation of proinflammatory cytokines and restriction of anti-inflammatory cytokines 72 . Sirt1 inhibition was found to promote Th9 cell differentiation and IL-9 production via the Sirt1-mTOR-HIF-1α axis 73 . In the same study, Wang et al. indicated that Sirt1-dependent regulation of glycolysis was critical for directing the differentiation of Th9 cells, highlighting the importance of metabolic reprogramming in controlling T cell fate.
The effects of Sirt1 on Th17 cells are controversial, and more studies are needed to dissect its precise role. A few studies have indicated that the differentiation of Th17 cells is affected by the degree of STAT3 deacetylation. Sirt1 activators such as metformin have been shown to impede Th17 cell differentiation and reduce IL-17A and RORγt expression via deacetylation of the STAT3 transcription factor. STAT3 deacetylation restricts its ability to translocate into the nucleus to induce RORγt transcription 74 . Another study showed that in vivo activation of Sirt1 using NAD + supplementation delayed the onset of experimental autoimmune encephalomyelitis (EAE). This protection was hypothesized to be a result of the decrease in Th17-mediated inflammatory responses induced by enhanced Sirt1 expression 75 . Moreover, treatment with methylene blue, another Sirt1 activator, alleviated Th17 responses and significantly reduced the clinical scores of EAE in mice 76 . In contrast, other researchers have shown that Sirt1 activation promotes the Th17 phenotype via RORγt deacetylation. Deacetylated RORγt appears to have stronger transcriptional Fig. 2 The subcellular localization and main functions of the mammalian sirtuins. Sirt1 is predominantly located in the nucleus and can also be found in the cytosol. Sirt2 is predominantly localized in the cytosol but can shuttle to the nucleus. Sirt3, Sirt4, and Sirt5 are mitochondrial proteins. Sirt6 and Sirt7 are localized in the nucleus and nucleolus, respectively. The main cellular functions are indicated in the boxes.
activity than Foxp3, thus strengthening the Th17 proinflammatory phenotype and suggesting that Sirt1 inhibitors may protect against autoimmune diseases 77 .
Foxp3 is a master regulator of Treg cell development and function and has three lysine acetylation sites (K31, K262, and K267) targeted by Sirt1 78 . Hyperacetylation of Foxp3 prevents its polyubiquitination and proteasomal degradation. Accordingly, Sirt1 deacetylase activity has been found to reduce Foxp3 protein levels, and treatment with Sirt1 inhibitors results in increased functional Treg cells 79 . In addition to the posttranslational control of Foxp3 by Sirt1, some studies reported that genetic deletion or pharmacologic inhibition of Sirt1 increased the number and suppressive activity of Foxp3 + Treg cells by increasing Foxp3 mRNA levels 78,79 . In contrast, Sirt1 was reported to promote Treg cell survival by stabilizing the Notch1 intracellular domain proximal to the membrane, given that the Notch receptor is essential for Treg cell survival within caloric-restricted conditions 80 .
Basic leucine-zipper ATF-like transcription factor (BATF) regulates multiple aspects of the T cell immune response 81 . Sirt1 was reported to impact CD8 + T cell differentiation and effector functions under the influence of BATF. Indeed, BATF has been shown to transcriptionally inhibit Sirt1 expression, resulting in increased histone acetylation at the T-bet locus. In turn, high levels of T-bet expression promote CD8 + T cell differentiation, and loss of BATF consequently inhibits CD8 + T cell differentiation 82 .
The metabolic switch toward FAO is a characteristic of T M cell differentiation, and this switch is known to be supported by the transcriptional coactivator PGC-1, which is involved in mitochondrial biogenesis and OxPhos metabolism 83 . Sirt1-mediated deacetylation has been shown to increase the transcriptional activity of the PGC-1α and PGC-1β cofactors 84,85 , suggesting a potential role for Sirt1 in promoting T M cell formation. A previous report indicated a decrease in Sirt1 expression levels in terminally differentiated CD8 + CD28 − T M cells, which concomitantly displayed enhanced glycolytic and cytotoxic capabilities 86 , suggesting that Sirt1 may restrain glycolytic metabolism in T cells (Fig. 3).

SIRT2
Until recently, our knowledge on the role of Sirt2 within the immune system was limited to its anti-inflammatory function via negative regulation of the NF-κB p65 subunit 87 . Accordingly, Sirt2deficient mice were found to develop severe forms of dextran sodium sulfate (DSS)-induced colitis via polarization of bone marrow-derived macrophages 88 . Furthermore, that study observed a greater proportion of activated CD4 + CD69 + T cells in the mesenteric lymph nodes of Sirt2 −/− mice in response to DSS-induced colitis, suggesting a role for Sirt2 in limiting CD4 + T EFF cell functions 88 .
A recent publication uncovered a new function of Sirt2 as a master regulator of T cell metabolism and functions. Hamaidi et al. found increased proliferation of CD4 + and CD8 + Sirt2 −/− T cells upon TCR activation and activated CD8 + T cells displayed increased IFN-γ production and granzyme B expression, leading to superior cytotoxic activity. This hyperreactive phenotype of Sirt2 −/− T cells endowed mice with superior antitumor immunity upon tumor challenge in vivo 89 .
Mechanistically, this hyperactivation and enhanced function of Sirt2 −/− T cells is linked to Sirt2 regulation of multiple metabolic pathways that play crucial roles in T cell effector functions. Sirt2 deacetylates and negatively impacts the activity of key metabolic enzymes of the glycolysis, TCA cycle, FAO and glutaminolysis pathways. Accordingly, Sirt2 deletion during T cell activation was followed by hyperacetylation of multiple metabolic enzymes and amplification of their activities, leading to increased aerobic glycolysis, OxPhos, FAO, and glutaminolysis (Fig. 3) 89 .
Sirt2 was found upregulated in T M cell stages. T M cells rely on FAO metabolism, and Sirt2 negatively impacts FA catabolism; thus, Sirt2 deletion promoted CD4 + and CD8 + T M cell formation ex vivo, which was associated with superior cell survival and decreased apoptosis. Consistently, increased accumulation of T M cells within the TME and the secondary lymphoid organs of Sirt2 −/− mice was observed upon tumor challenge, whereas no qualitative or quantitative phenotypic differences were observed between unchallenged WT and Sirt2 −/− control mice 89 .
That study investigated the role of Sirt2 during T cell activation and maturation and within the antitumor immune response 89 . However, the role of Sirt2 in different CD4 + T cell subsets with distinct metabolic programs, including Treg, Th1, Th2, Th9 and Th17 cell subsets, requires further investigation.

SIRT3
Sirt3 localizes mainly to the mitochondrial matrix and plays an important role in regulating elements of mitochondrial metabolism, including the TCA cycle, urea cycle, FAO, and reactive oxygen species (ROS) detoxification (Fig. 3) 90 . Sirt3 promotes energy Fig. 3 Mammalian sirtuins and metabolic reprogramming. The entry of glucose into the cell is mediated by the glucose transporter Glut1. Glucose is then metabolized to pyruvate, which enters the mitochondrial tricarboxylic acid (TCA) cycle and generates reducing equivalents for ATP production via oxidative phosphorylation (OxPhos). During aerobic glycolysis, pyruvate is fermented into lactate in the cytoplasm despite the availability of oxygen for complete glucose oxidation; this process is called the Warburg effect. Glutamine enters cells using alanine, serine, cysteinepreferring transporter 2 (ASCT2), and it is converted into glutamate by glutaminase (GLS) and into α-ketoglutarate (α-KG) by glutamate dehydrogenase (GDH), which enters the TCA cycle for ATP production via OxPhos. Fatty acid molecules are catabolized into acetyl-CoA via fatty acid oxidation (FAO), and acetyl-CoA enters the TCA cycle for ATP production. Sirtuins are metabolic sensors that modulate a variety of metabolic pathways. Sirt1, Sirt3, and Sirt6 restrain the glycolytic pathway through hypoxia-inducible factor 1-alpha (HIF-1α) inhibition or direct effects. Sirt3 upregulates the OxPhos pathway by enhancing the activity of mitochondrial complexes I, II, and III and dampening reactive oxygen species (ROS) production. Sirt1 is also able to increase FAO by activating PPAR-α and PGC1, while Sirt3 upregulates FAO under caloric restriction conditions. Sirt2 deacetylates and inhibits the activity of many metabolic enzymes involved in glycolysis, glutaminolysis, the TCA cycle and FAO. Sirt3 and Sirt4 activate and inhibit glutaminolysis, respectively, by regulating GDH activity. Sirt6 impacts the glutaminolysis pathway by regulating c-Myc transcriptional activity. Sirt5 increases glycolysis by increasing the activity of the glycolytic enzyme GAPDH. Sirt7 can also repress HIF-1α and, therefore, may inhibit the transcription of glycolytic genes. generation; thus, its expression is higher in metabolically active tissues.
A recent report indicated that T cells from Sirt3-deficient (Sirt3 −/− ) donor mice caused reduced graft-versus-host disease (GVHD) severity in comparison to T cells from control donor mice, suggesting that Sirt3 targeting can improve transplant recipient outcome. In that study, the protective effect of allogeneic Sirt3 −/− T cells was related to reduced T cell proliferation and CXCR3 expression, with no significant impact on cytokine secretion or cytotoxic functions 91 .
Furthermore, Sirt3 deficiency in mouse models had no impact on immune responses against bacterial and fungal infections 92 , suggesting that Sirt3 may play a limited role in T EFF cell functions.
Inhibition of OxPhos impairs Treg cell function, and given the key role of Sirt3 in OxPhos, it is expected that Sirt3 promotes the suppressive activity of Treg cells. In fact, Treg cells from Sirt3 −/− mice exhibited impaired suppressive functions, as demonstrated in in vitro suppression assay and an in vivo allograft model, and HDAC9 deletion was found to increase Treg suppressive activity by increasing Sirt3 expression 93 .
Sirt3 is also involved in CD8 + T cell function. Sirt3 −/− CD8 + T cells exhibited reduced ROS production and CXCR3 expression upon activation. Moreover, T cells from Sirt3 −/− donor mice were able to reduce GVHD within the gastrointestinal tract, which is probably due to decreased CXCR3-dependent CD8 + T cell trafficking to the site 93 .

SIRT4
Sirt4 is a mitochondrial sirtuin with ADP-ribosylation activity. No deacetylase activity of Sirt4 has yet been identified. By ADPribosylating glutamate dehydrogenase (GDH), Sirt4 represses its enzymatic activity, which limits the metabolism of glutamine into glutamate to generate ATP (Fig. 3) 94 . It is thus conceivable that T cell metabolic activity and effector functions can be enhanced by Sirt4 inhibition via increasing glutaminolysis, another ATPgenerating pathway used by activated T cells. Furthermore, given the role of glutaminolysis in Th17 cell differentiation 95 and Treg cell development 18,19 , we would also expect a proinflammatory phenotype with Sirt4 deficiency. Sirt4 was found to physiologically break immune tolerance and to resolve acute inflammation in a model of sepsis by coordinately reprogramming the metabolism and bioenergetics of human monocytes 96 .
Recently, in a mouse model of neuroinflammation following traumatic spinal cord injury, Sirt4 expression was found to be upregulated in infiltrating Treg cells in the spinal cord parenchyma 97 . Interestingly, Sirt4 overexpression in splenic naive Treg cells was found to alleviate the expression of Foxp3, IL-10, and TGF-β and to weaken the inhibitory activity of Treg cells. Additionally, Sirt4 overexpression blocked in vitro inducible Treg cell generation from conventional T cells 97 . Consistently, Sirt4 knockdown increased the anti-inflammatory activity of infiltrating Treg cells in the parenchyma of injured spinal cords 97 . The authors concluded that Sirt4 inhibits the anti-neuroinflammatory activity of Treg cells by blocking AMPK signaling given that an AMPK agonist restored the expression of Foxp3 and IL-10 in Treg cells 97 . However, this result could also be related to Sirt4's impact on glutamine metabolism and consequently on Treg cell development.

SIRT5
Sirt5 is another mitochondrial sirtuin that displays a weak deacetylase activity. However, Sirt5 is unique in executing novel enzymatic activities involving lysine desuccinylation, demalonylation, and deglutarylation 98 . Given the emerging role of protein succinylation in the immune response 99 , future studies focusing on the role of Sirt5 in T cell function are needed.
An anti-inflammatory protective role of Sirt5 was reported in a DSS-induced colitis mouse model. Sirt5 was found to suppress IL-1β production and proinflammatory responses in macrophages by regulating pyruvate kinase M2 (PKM2) succinylation. Sirt5dependent succinylation promotes PKM2 entry into the nucleus, where the PKM2-HIF1α complex is formed at the promoter of the IL-1β gene to stimulate its transcription 100 . Paradoxically, proinflammatory activity of Sirt5 was described during the acute and immunosuppressive phases of sepsis. The induction and persistence of a hypoinflammatory and immunosuppressive state in severe sepsis are commonly associated with increased risks of secondary infections and mortality. Sirt5 was found to rescue the innate inflammatory response of endotoxin-tolerant macrophages by promoting acetylation of NF-κB p65. Mechanistically, Sirt5 competes with Sirt2 to interact with NF-κB p65 and block its deacetylation by Sirt2, which consequently leads to increased acetylation of p65 and activation of the NF-κB pathway with its downstream cytokines 101 .
Although little work has been done to study Sirt5 in the adaptive immune response, a recent study has demonstrated a pivotal role of Sirt5 in regulating T cell activation and differentiation 102 . Indeed, Sirt5 deficiency was found to promote mouse naive T cell activation and increased IFN-γ production upon TCR ligation and to further influence T cell differentiation. Sirt5 deletion enhanced Th1 cell and CTL differentiation and decreased CD4 + Treg cell differentiation, whereas no qualitative or quantitative phenotypic differences were observed in the peripheral lymphoid organs of WT and Sirt5 −/− mice under steady state conditions 102 . More importantly, even though Sirt5 −/− mice were found to be highly susceptible to DSS-induced colitis 100 , Sirt5 −/− mice were resistant to colorectal tumorigenesis following experimentally induced colitis, and this resistance was related to increased IFN-γ production in the colon tissue by immune cells 102 . While these data indicate the importance of Sirt5 in T cell activation and antitumor functions, the molecular mechanisms of Sirt5 activity need to be further elucidated.
Remarkably, studies on a large panel of preclinical mouse models of sepsis showed that Sirt5 deficiency has no impact on antimicrobial host immune defenses 103 , suggesting that Sirt5 plays a limited role in T EFF cell functions. On the other hand, these observations support the assumption that therapies directed against Sirt5 do not impair antibacterial host defenses.
Recent studies have revealed that Sirt6 possesses antiinflammatory properties. Sirt6 represses proinflammatory gene transcription by affecting chromatin structure rather than by directly deacetylating NF-κB, while Sirt1 and Sirt2 directly deacetylate p65 and inhibit its transcriptional activity 87,106 . Although Sirt6 physically associates with the p65 subunit, it modulates its transcriptional activity by deacetylating H3K9 on the promoter of selected NF-κB target genes 107 . Sirt6 was also reported to interact with JUN and deacetylate H3K9 at the promoter of proinflammatory genes whose expression involves JUN. The same study also indicated that Sirt6-null mice develop chronic liver inflammation attributable to Sirt6 deficiency in immune cells and macrophages, and these cells express increased levels of MCP1, IL-6, and TNF 108 . While Sirt6 appears to exert antiinflammatory action when acting at the transcriptional level, scattered evidence seems to suggest that Sirt6 induces proinflammatory effects by modulating distinct intracellular signaling factors, such as Ca2 + homeostasis 109 and mRNA translation 110 . Indeed, Sirt6 has been shown to promote the expression of cytokines and chemokines, including IL-6, TNF, CXCL2, and IFN-γ, and to positively regulate TNF secretion by increasing mRNA translational efficiency in several immune cells [110][111][112] .
Little is known about the role of Sirt6 in the adaptive immune response. HIF-1α is a transcriptional regulator of glycolysis and is known to regulate the production of several cytokines 113 and to enhance Th17 cell differentiation while concomitantly inhibiting Treg cell function 114,115 . Furthermore, Myc is a global regulator of the immune response and plays a crucial role in glutamine metabolism 15 . Glycolysis and glutaminolysis are critical metabolic pathways on which T EFF cells depend to meet their energy needs. Dynamic switching between these metabolic pathways needs to be tightly regulated to achieve optimal function within immune cells.
Sirt6 appears to strongly oppose glycolysis by inhibiting the transcription of many glycolytic genes 116 . Sirt6 has been shown to physically interact with HIF-1α and to corepress its transcriptional activity by deacetylating histone H3K9 at the promoters of several glycolytic genes 117 . Furthermore, Sirt6 also regulates glutaminase expression, thus modulating the glutaminolysis pathway. Sirt6 interacts with Myc and reduces its transcriptional activity by deacetylating histones at the promoter region of Myc target genes 118 . Collectively, these observations suggest that Sirt6 potentially has an immunomodulatory effect on T cell metabolism and functions by regulating the HIF-1α and Myc pathways (Fig. 3).

SIRT7
Sirt7 is the last and least studied member of sirtuins. Sirt7 is a vital regulator of ribosome biogenesis, and it is enriched in nucleoli, where it facilitates RNA polymerase I-dependent transcription of ribosomal (r)RNA genes 119 . Sirt7 expression is linked to cell proliferation and oncogenic activity, connecting Sirt7-dependent regulation of ribosome biogenesis with cell cycle progression, metabolic homeostasis, stress resistance, aging and tumorigenesis [120][121][122] .
Recent studies suggest that Sirt7 is also involved in inflammation. Vakhrusheva et al. showed that Sirt7 deletion predisposes mice to heart hypertrophy and cardiac inflammation. These authors observed an increased infiltration of immune cells, with higher levels of proinflammatory cytokine production 123 . Information regarding the possible involvement of Sirt7 in the adaptive immune response is currently very limited.

CONCLUDING REMARKS AND FUTURE PERSPECTIVES
The well-documented capacity of sirtuins to promote the resolution of the inflammatory response has led many research groups to focus on developing therapeutic strategies to target their enzymatic activity in vivo. However, to achieve the desired clinical outcomes, it is paramount to decide whether to stimulate or block sirtuin activity and how to modulate a specific sirtuin within a specific T cell subset. All these challenges are worthy of further study due to the ubiquity of sirtuins. In addition, these proteins have intricate and partially unknown functions within an organism, reflecting the immense challenge of targeting any of them.
For example, enhancing Treg cell suppressive activity with Sirt3 agonists to attenuate inflammation represents a potential strategy to treat autoimmunity. Similarly, the use of Sirt1 activators to suppress inflammation could help treat autoimmune diseases. Metformin, classically prescribed for the management of type 2 diabetes, appears to have a stimulatory effect on sirtuins. Sirt1 activation by metformin has been shown to decrease inflammation by decreasing Th17 cell differentiation 74 . Resveratrol, a compound with sirtuin-activating effects, is currently of intense interest within both scientific and lay communities for its anti-inflammatory properties 124 . Resveratrol stimulates Sirt1 activity, which decreases c-Jun acetylation, consequently restraining T cell activation 62 . Resveratrol has been shown to improve outcomes in several experimental autoimmunity models 125 . For instance, administration of resveratrol protected mice against experimental rheumatoid arthritis by inhibiting Th17 cell differentiation 126 . Furthermore, resveratrol was proven to have both preventative and therapeutic effects by reverting the advanced stages of insulitis in a nonobese diabetic (NOD) mouse model due to reduced Th17 cells 127 . Moreover, resveratrol induced protective effects against high-fat diet-induced obesity in mice in part by increasing total Treg cells 128 .
Despite the obvious progress in the organ transplantation field, the commonly required immunosuppressive therapies are known to ultimately induce cancer and quite often infections. Presently, sirtuins are being thoroughly studied for their role in promoting organ transplant tolerance. Treg and Th17 cells drive transplant tolerance, and in relation to these T cell subsets, Sirt1 has emerged as an eligible target for clinical interventions in transplantation. Indeed, Sirt1 deletion improved Treg cell suppressive activity in vitro and in vivo, and mice with specific Sirt1 deletion in Foxp3 + Treg cells exhibited prolonged survival after cardiac allografts concomitant with increased Treg cell infiltration. In the same study, similar results were obtained when using Sirt1 inhibitors in vivo 129 . Later, comparable findings were reported after kidney transplantation from BALB/c mice into C57BL/6 recipient mice treated with EX-527, a Sirt1 inhibitor 130 .
Recently, the effect of sirtinol, a Sirt1 inhibitor, on heart allograft survival was evaluated, and it was found that sirtinol-treated recipients exhibited increased numbers of Foxp3 Treg cells and reduced numbers of Th17 cells in addition to decreased expression of the IL-17A and RORγt transcription factor 131 . Therefore, Sirt1 inhibition might affect the Th17/Treg cell ratio in favor of Treg cells. In contrast, Sirt1 activators such as metformin were found to decrease IL-17A and RORγt expression in mouse models as described earlier 74 . Certainly, more studies are required to define the appropriate action of Sirt1 targeting for allograft retention.
To date, the research community has focused on promoting the anti-inflammatory activity of sirtuins to treat a multitude of autoimmune diseases and other chronic diseases associated with inflammation. However, we must extend our interest toward blocking the activity of sirtuins to promote the effector activity of T cells in the context of antitumor immunity. Indeed, enhancing tumor rejection by boosting tumor-reactive T cell activity would be tremendously effective in fighting cancer. Recent reports demonstrating the immunotherapeutic potential of Sirt2 and Sirt5 blockade in T cells to reject tumor challenge 89 and to protect against colorectal tumorigenesis in murine models, highlight the expected effectiveness of such strategy 102 . In addition, sirtuins have been shown to play an oncogenic role in many tumor types 132 , and sirtuin inhibitor drugs have been shown to have tumor-suppressive activity on cancer cells, suggesting an intriguing possibility that in certain cancer patients, targeting sirtuins may suppress tumor proliferation while simultaneously boosting antitumor immunity. Alternatively, manipulation of sirtuins could be achieved by gene editing during TIL expansion or chimeric antigen receptor (CAR) T cell generation ex vivo, thus selectively targeting T cells in the context of immune cell therapy 133,134 .
Many reports indicate that exhausted tumor-infiltrating lymphocytes display dysregulated metabolism within the metabolically restricted tumor microenvironment 22,23,135,136 . Modulation of sirtuins to enhance the metabolic activity of T cells could provide a viable strategy to restore exhausted tumor-reactive T cells.


extfieldsofstudy: Medicine Biology

text:
High carriage of plasmid-mediated quinolone resistance (PMQR) genes by cefotaxime-resistant Escherichia coli recovered from surface-leaking sanitary sewers

There is a rapid rise in the incidence of quinolone resistant bacteria in Nigeria. Most studies in Nigeria have focused on isolates from the clinical settings, with few focusing on isolates of environmental origin. This study aimed to investigate the antibiogram and carriage of plasmid-mediated quinolone resistance (PMQR) genes by quinolone-resistant isolates obtained from a pool of cefotaxime-resistant Escherichia coli (E. coli) recovered from sewage leaking out of some surface-leaking sanitary sewers in a University community in Nigeria. Isolation of E. coli from the sewage samples was done on CHROMagar E. coli, after enrichment of the samples was done in Brain Heart Infusion broth amended with 6 µg/mL of cefotaxime. Identification of presumptive E. coli was done using molecular methods (detection of uidA gene), while susceptibility to antibiotics was carried out using the disc diffusion method. Detection of PMQR genes (qnrA, qnrB, qnrS, aac(6')-lb-cr, qepA and oqxAB) was carried out using primer-specific PCR. A total of 32 non-repetitive cefotaxime-resistant E. coli were obtained from the sewage, with 21 being quinolone-resistant. The quinolone-resistant isolates showed varying level of resistance to the tested antibiotics, with imipenem being the only exception with 0% resistance. The PMQR genes: aac(6')-lb-cr, qnrA, qnrB, qnrS and qepA and oqxAB were detected in 90.5%, 61.9%, 47.6%, 38.1%, 4.8% and 0% respectively of the isolates. The findings of this study showed a high level of resistance to antibiotics and carriage of PMQR genes by quinolone-resistant E. coli obtained from the leaking sanitary sewers, suggesting a potential environmental and public health concern.

Introduction
The wastewater environment, specifically composed of high concentrations of bacteria, nutrients, and suspended solids, is a known potential hub for the inception of antibiotic resistance and the stimulation of horizontal gene transfer (Lorenz and Wackernagel 1994). The high levels of antibiotics typically discharged in human wastes into the environment, is known to sufficiently inhibit susceptible bacteria and provide a selective advantage to the resistant ones (Kummerer 2001), making sewage and other wastewaters a favorable environment for bacterial growth and the likely spread of antibiotic resistance (Mezrioui and Baleux 1994). The typical end products (effluent or bio-solids), or non-typical routes like seepage through cracks in tanks, flooding or animal contact, and also aerosols, are channels through which bacteria in wastewater may enter into the environment (Brandi et al. 2000).
The use of quinolone antibiotics in human and veterinary medicine is on the rise due to their efficacy as important weapons against pathogenic bacteria via their ability to selectively inhibit the synthesis of DNA. This group of antibiotics constitutes about 17% of the global consumption of antibiotics (Van Doorslaer et al. 2014), and are frequently encountered in domestic and hospital sewage, because they are largely excreted unchanged in urine and faecal materials. (Kaplan et al. 2013). Quinolones are not easily degraded, and are adsorbed to sewage sludge during biological treatment (Kümmerer 2009). In a study carried out by Lindberg et al. (2006) and Fink et al. (2012), they found out that fluoroquinolones are hardly removed in effluent treatment plants, with approximately 70% remaining attached to sludge. In addition to this, the low effectiveness of treatment plants cannot be overlooked in the continuous spread of multidrug resistant (MDR) bacteria into water aquifers (Korzeniewska and Harnisz 2013).
The occurrence of quinolones in the environment has caused some substantial effects on the microbial structure and community, due to the exposure of these flora to the antibiotics at sub-lethal concentration, making them develop resistance to these agents. This eventually makes the environment a repository of antibiotic resistant bacteria carrying several resistance genes and other mobile genetic elements (MGE) (Aminov 2009;Cordova-Kreylos and Scow 2007).
The detection of PMQR determinants in the late 1990s has opened the doors of research into the mechanisms of quinolone resistance in bacteria and their transferability. Different resistance mechanisms have so far been described and they range from the qnr gene families which encode proteins that protect DNA gyrase and topoisomerase IV from the destructive action of quinolones; aac(6′)-Ib-cr gene, coding for aminoglycoside acetyltransferase, which are involved in the acetylation of fluoroquinolones such as ciprofloxacin and norfloxacin; and two active efflux pumps, qepA and oqxAB, which confer decreased susceptibility to fluoroquinolones (Poirel et al. 2012;Ruiz et al. 2012).
Most data on quinolone resistance in Nigeria have focused majorly on the occurrence of these genes in clinical isolates, therefore making data related to the environment very few. This study therefore aimed at investigating the carriage of PMQR genes by cefotaxime-resistant Escherichia coli isolated from surface-leaking sanitary sewers within a University community.

Study site and sample collection
The study was carried out within the environment of an institution of higher learning in South-west Nigeria.
Sanitary wastes from the students' hall of residence, staff quarters, other residential areas and the business pools are being channeled to a treatment plant via sanitary sewers located underground all across the University premises. A common challenge with this system of waste transportation is the breakage of conveyor pipes as a result of several factors. This often times leads to the outflow of the content of the pipes into the environment, creating a very horrible sight and foul-smelling odour. Although this situation is remedied by the concerned unit of the University, it takes time before a positive action is taken on such breakage. The horrible sight of such breakage was a major motivating factor that stimulated the execution of this study. Sewage from the burst pipes were collected aseptically at different dripping points and transported to the laboratories on ice for microbiological analysis. A total of eight sewage samples were collected at the earmarked dripping points on the sewers. Samples were analyzed about one hour after collection.

Isolation of cefotaxime-resistant Escherichia coli from the samples
Aliquot (2 mL) of each sewage sample was inoculated into 3 mL Brain Heart Infusion broth (Becton, Dickinson and Company, USA), to which a disc of cefotaxime (30 µg), purchased from Oxoid, UK, has been added, giving a final concentration of 6 µg of the antibiotic in each tube. The tubes were incubated overnight at 35 ± 2 °C. Using the streak plate technique, a loopful of each of the cefotaxime-amended samples was inoculated onto the surface of already prepared plates of CHROMagar E. coli (CHROMagar, France) and incubated overnight at 35 ± 2 °C. Blue colonies presumptive of Escherichia coli were picked, purified and stored in glycerol stock (15% glycerol).

Identification of cefotaxime-resistant Escherichia coli
The genomic DNA of presumptive Escherichia coli was extracted using the boiling lysis method according to the modified method of Gugliandolo et al. (2010). The identity of the isolates was confirmed by detecting the presence of uidA, a housekeeping gene in Escherichia coli. The method of Janezic et al. (2013) was used for the PCR amplification of the uidA gene. Escherichia coli OG1a possessing the uidA gene as reported by Adekanmbi et al. (2020) was used as the positive control. Isolates possessing the uidA gene were selected for the antibiotic susceptibility testing and detection of PMQR genes. The details of the uidA primers are shown in Table 1.

Susceptibility to antibiotics
The confirmed Escherichia coli were subjected to a panel of eight antibiotics using the Kirby-Bauer disc diffusion method (Kirby-Bauer 1996). The procedures used for the susceptibility testing, including the choice of medium, inoculum standardization, reading and interpretation of the zone diameter were as described by CLSI (2018). Isolates that were resistant to ciprofloxacin (a fluoroquinolone), were selected for the detection of the PMQR genes which encode quinolone resistance.

Detection of PMQR genes
The detection of six genes encoding PMQR in confirmed ciprofloxacin-resistant (CIP-resistant) Escherichia coli was done using the methods of Wu et al. (2007) and Park et al. (2009). The detection of qnrA, qnrB and qnrS and qepA was carried out using a duplex PCR, while a monoplex PCR was used for the detection of aac(6')-Ib-cr and oqxAB. The PCR reaction volume was 25 µL consisting of 5 × PCR Master Mix (Jena Bioscience, Germany), forward and reverse primers (0.25 µL each, DNA template (1 µL), with PCR quality water making up the volume. The oligonucleotide primers used in this study are highlighted in Table 1.

Results
Thirty-two non-repetitive cefotaxime-resistant Escherichia coli isolates were recovered from the surface-leaking sanitary sewers, with all the eight collected samples showing the presence of the organism. Twenty-one of the isolates representing 65.6% of the total isolates were resistant to ciprofloxacin (CIP), while 34.4% (11/32) were sensitive to the antibiotic.
The susceptibility of the isolates to a panel of eight antibiotics is highlighted in Table 2. The observed resistance by the isolates to the antibiotics varied, with the only exception being imipenem, to which no resistance was observed. Six of the isolates (28.6%) showed resistance to amoxicillinclavulanate, seven (33.3%) were resistant to ceftazidime, 10 (47.6%) showed resistance to gentamicin and 19 (90.5%) were resistant to trimethoprim/sulfamethoxazole. There was 100% resistance to each of cefotaxime, cefpodoxime and cefuroxime.  All the targeted PMQR genes were detected in varying number of isolates, with the notable exception being oqxAB gene, which was not detected at all in any isolate as shown in Tables 3 and 4. Of the six PMQR genes targeted in this study, qnrA was detected in 13 (61.9%) of the isolates, making it the second most prevalent PMQR gene, after aac(6')lb-cr (which was the most prevalent) with 19 (90.5%) of the isolates harbouring it. The frequency of occurrence of qnrB, qnrS and qepA was 10 (47.6%), 8 (38.1%) and 1 (4.8%) respectively. oqxAB was not detected in any of the 21 CIP-resistant Escherichia coli. One of the isolates (E. coli SWS2a), did not carry any of the six PMQR genes targeted, while six isolates carried one PMQR gene only. Four isolates carried two and three PMQR genes respectively, while four of the six PMQR genes were carried by 6 (28.6%) of the isolates. There was co-occurrence of PMQR genes in 14 (66.7%) of the isolates.

Discussion
The fluoroquinolone antibiotics have been used extensively in the treatment of human and animal infections, mainly because of their selective inhibition of the synthesis of DNA in bacterial cells. They account for close to one-fifth of the global antibiotic consumption (Van Doorslaer et al. 2014). Ciprofloxacin, a second-generation fluoroquinolone has been widely used in the treatment of bacterial infections of human origin and is the one of the most abundant antibiotics detected in municipal biosolids, owing to their widespread use and the fact that it is excreted mostly unchanged in urine and wastes of faecal origin, making them available at high concentration in wastewater from sewage and clinical settings (Kaplan et al. 2013;Van Doorslaer et al. 2014;Xiong et al. 2015). The major difference between the quinolones of the first and second generations is the possession of a fluorine atom at the sixth carbon atom of the quinolone moiety by the latter, and they exhibit a high level of potency against gram-negative bacteria and several gram-positive bacterial genera. In addition to this, however, ciprofloxacin shows a high level of activity against Pseudomonas aeruginosa,  making it a drug of choice in the treatment of a variety of infections (Panton and Reeves 1988;Van Bambeke et al. 2005).
Resistance to fluoroquinolones has increased worldwide over the past few years and this has limited available treatment options, resulting in several cases of resistance and treatment failures (Briales et al. 2012;Okade et al. 2014;Piekarska et al. 2015). The rising tide of antimicrobial resistance has been majorly linked to the widespread and continuous use of antibiotics in agriculture, human medicine and veterinary practices. This has placed a lot of selective pressure on bacteria, making it difficult to curtain the geometric rise in antibiotic resistance over the years (Baquero et al. 2008;Harnisz et al. 2015;Kotlarska et al. 2015). Sewage and wastewater on the other hand have been taunted as ideal environment for the acquisition of antibiotic resistance genes (ARG) and this has been largely linked to influence from anthropogenic activities (Marti et al. 2014;Osińska et al. 2016).
In our study, a total of 21 CIP-resistant E. coli was obtained from the pool of 32 cefotaxime-resistant isolates recovered from the broken sanitary sewers. Bacteria showing resistance to quinolones, especially E. coli have been well reported in several studies. Osińska et al. (2016) reported the isolation of fluoroquinolone-resistant bacteria from sewage and surface water samples in Poland, while studies carried out by Guillard et al. (2014), Yang et al. (2014) and Ranjbar et al. (2019), pointed out the fact that Escherichia coli ranked as the most frequently occurring fluoroquinoloneresistant bacteria from water, wastewater, animals, food and the clinical environment. A study carried out in Nigeria by Ehwarieme and his colleagues reported the occurrence of fluoroquinolone-resistant enteric bacteria from samples of human and animal origin, with Escherichia coli being the predominant organism obtained (Ehwarieme et al. 2021).
In this study, the target PMQR genes with the exception of oqxAB, were detected in 20 of the 21 CIP-resistant isolates obtained, with one isolate devoid of any of the six PMQR genes. The qnr determinants, which have been reported to occur on chromosomal DNA in many environmental strains (Poirel et al. 2005) are considered exclusively plasmid-borne in the Enterobacteriaceae (Rodriguez-Martinez et al. 2011). These genes are proteins of the pentapeptide repeat family and are concerned with the protection of DNA gyrase and topoisomerase (IV) from the deleterious effect of the quinolone antibiotics. They are a very diverse group of PMQR determinants and have been found resident in bacteria of animal, human and environmental origin. In this current study, three variants of the qnr gene were targeted, with varying degree of detection observed in the CIP-resistant isolates. Of the qnr determinants, qnrA was the most prevalent in this study, with 13 of the isolates (61.9%) harbouring it, followed by qnrB with 10 isolates (47.6%) and qnrS 8 (38.1%). The relative occurrence of the qnr genes in this study is higher than the frequency of occurrence in a study carried out by Ranjbar et al. (2019), where they reported the detection of qnrA, qnrB and qnrS in 1%, 9% and 28% of the total isolates obtained from water sources in Iran, while a study on hospital wastewater in the same country by Ranjbar et al. (2017), reported the detection of qnrB and qnrS in eleven (45.8%) and seven (29.2%) respectively of the E. coli isolates obtained. However, none of the isolates in their study harboured qnrA gene, which is contrary to this study.
The quinolone efflux pump (qepA) which was first described in a study by Yamane et al. (2007), confers on the bacterial cell, the ability to reduce quinolone accumulation in the cell. It has been detected widely in many gram-negative genera in most countries of the world. In our study, one isolate (4.8%) of the entire 21 CIP-resistant bacteria, harboured this particular gene. A low frequency of occurrence of the gene has been reported in several studies, and this could be linked to its limited host spectrum. Chen et al. (2007) reported a percentage occurrence of 2.6% for qepA in 1022 E. coli isolates obtained in their study, while Cattoir et al. (2007) and Yamane et al. (2008) detected qepA in 0.8% and 0.3% of the total isolates obtained in their respective studies.
There is limited data on oqxAB, an efflux pump-mediating gene, which was discovered in the late 2000s. Its role as a PMQR determinant was not identified until then, thereby making data available on the gene quite limited. It confers resistance to the veterinary growth-promoting quinoxaline-di-N-oxide olaquindox, and was first discovered in Escherichia coli from swine (Hansen et al. 2004;Sørensen et al. 2003;Strahilevitz et al. 2013). The frequency of occurrence of the gene in most studies is very low in comparison with other PMQR determinants and this could explain the absence of the gene in all the CIPresistant isolates obtained in this study.
Aminoglycoside acetyltransferase (aac(6')-lb-cr) acts by causing the acetylation of quinolones, causing the modification of the antibiotics, which eventually leads to their inactivation. On its own, the resistance conferred on bacteria by aminoglycoside acetyltransferase is typically low, but it becomes elevated with the presence of qnr gene in the cell. In most reports on quinolone resistance in bacteria, aac(6')-lb-cr appears to be the commonest PMQR determinant (De Jong et al. 2018;Strahilevitz et al. 2009;Shaheen et al. 2013), and this corroborates the observation in this study, where 19 of 21 (90%) of the CIP-resistant bacteria carried the gene. This observation is in agreement with several other reports where the occurrence of the gene and its high prevalence in clinical samples and wastewater of hospital origin have been widely reported. In a study on hospital wastewater carried out by Chandran et al. (2014), they detected aac(6')-lb-cr in 89% of their isolates, while Guillard et al. (2014) and Piekarska et al. (2015) detected the gene in 96.7% (118/122) and 85.7% of isolates obtained from samples and wastewater of clinical origin, respectively in their studies. In a different twist to the aforementioned, Su et al. (2013) reported the detection of aac(6')-lb-cr in 5.1% of the total 452 quinoloneresistant isolates from a municipal treatment facility in China, making it the least occurring PMQR determinant in their study.

Limitations of the study
Despite the abundance of PMQR genes in the isolates obtained in this study, the limitations stem from the fact that the sampling process stopped as soon as the repair works on the burst sewers commenced, making it difficult to get more samples for the isolation of more resistant isolates. Despite this limitation, other studies could venture into the detection of PMQR determinants in other bacterial genera from the environment and the genomic characterization of such isolates using advanced molecular biology tools. This will enrich the few available reports on PMQR determinants in isolates of environmental origin.

Conclusion
This present study has provided some information on the occurrence of cefotaxime-resistant Escherichia coli carrying PMQR genes in sewage samples, with aac(6')-lb-cr being the most predominant PMQR gene, while oqxAB was not detected. The resistance shown to the tested antibiotics by the CIP-resistant isolates varied, with imipenem (a carbapenem) being the only antibiotic, to which resistance was not observed to. This suggests that the receiving environment of these untreated leaky wastewater is a potential repository of these resistant bacteria and PMQR genes, which could expose the University community to a potential public health challenge. This calls for a holistic approach to the concept of antibiotic resistance, which is majorly being looked at from the clinical perspective, as the environment is not spared of this menace. There is also a need for more proactive and urgent mechanisms to be put in place to address the issues of broken sewers within communities.


Body fat percentage is independently and inversely associated with serum antibody responses to SARS-CoV-2 mRNA vaccines

Vaccination is widely considered the most effective preventative strategy to protect against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. An individual’s exercise habits, and physical fitness have been shown to impact the immune response following vaccination using traditional vaccine platforms, but their effects are not well characterized following administration of newer vaccination technology (mRNA vaccines). We investigated these effects on the magnitude of antibody responses following SARS-CoV-2 mRNA vaccination while accounting for known covariates (age, sex, time since vaccination, and the type of vaccine administered). Adults of varying fitness levels (18–65 years; N = 50) who had received either the Moderna or Pfizer SARS-CoV-2 mRNA vaccine between 2 weeks and 6 months prior, completed health history and physical activity questionnaires, had their blood drawn, body composition, cardiorespiratory fitness, and strength assessed. Multiple linear regressions assessed the effect of percent body fat, hand grip strength, cardiorespiratory fitness, and physical activity levels on the magnitude of receptor binding domain protein (RBD) and spike protein subunit 1 (S1) and 2 (S2) while accounting for known covariates. Body fat percentage was inversely associated with the magnitude of S1 (p = 0.006, β = − 366.56), RBD (p = 0.003, β = − 249.30), and S2 (p = 0.106, β = − 190.08) antibodies present in the serum following SARS-CoV-2 mRNA vaccination. Given the increasing number of infections, variants, and the known waning effects of vaccination, future mRNA vaccinations such as boosters are encouraged to sustain immunity; reducing excess body fat may improve the efficacy of these vaccinations.

Scientific Reports
| (2022) 12:19224 | https://doi.org/10.1038/s41598-022-21884-z www.nature.com/scientificreports/ advent of multiple variants has contributed to continued SARS-CoV-2 infection, even among vaccinated and previously infected individuals 1,4 . To combat these variants, it is crucial to understand the wide inter-individual variability that alters the magnitude of protective immunity acquired after vaccination. The individual response to vaccinations varies substantially depending on the type of vaccine administered, age and sex of the individual, and time since vaccination 1,5 . For example, recent studies have demonstrated that protective immunity can wane within 6 months post vaccination increasing the risk of SARS-CoV-2 infection 6 . Importantly, modifiable factors related to one's lifestyle such as an individual's exercise habits, and physical fitness have also been shown to impact the immune response following vaccination. Researchers have demonstrated with previous vaccination programs that individuals who exercise regularly 7,8 , as well as those that performed an acute bout of exercise within 30 min of vaccination 9 , have improved antibody responses compared to their age-matched controls. Moreover, obesity is associated with impaired immune responses following the influenza vaccine [10][11][12] and COVID-19 vaccines 13 , whereas weight loss is associated with improved responses 14 . Together these findings demonstrate that levels of physical activity and body fat can affect the efficacy of vaccination. General lifestyle recommendations including regular exercise to support immune function amid the COVID-19 pandemic have been issued 15 , but the role of lifestyle factors on the prophylactic immune response to SARS-CoV-2 vaccination remains understudied 16 . A further element of uncertainty is that two prominent SARS-CoV-2 vaccines currently being distributed in the United States and other countries are mRNA-based vaccines 17 . Previous researchers have demonstrated that physical activity and obesity can contribute to heterogeneity of the immune response following vaccination with vaccines involving inactivated, attenuated, or recombinant viral protein vaccine platforms. Although a recent study showed that an acute bout of exercise after mRNA vaccination boosts antibody responses 9 , the effect of regular physical activity and body fatness on the antibody response following mRNA vaccination is not known. Additionally, the impact that other components of fitness such as muscular strength and cardiorespiratory (aerobic) fitness may have on prophylactic immune responses to mRNA SARS-CoV-2 vaccines are also unknown. Therefore, the purpose of this study was to address this gap in the literature by investigating the effects of exercise habits and several aspects of physical fitness on the magnitude of the antibody responses following SARS-CoV-2 mRNA vaccination. Given that previous researchers have shown that age, sex, time since vaccination, and the type of vaccine affect the antibody response to vaccinations 1,5 , the current study aimed to evaluate the effect of exercise habits and components of physical fitness on the antibody response following vaccination at the individual level while accounting for these covariates.

Materials and methods
Experimental design. Using a cross-sectional study design, participants who had received their second dose of either the Moderna or Pfizer SARS-CoV-2 mRNA vaccine between 2 weeks and 6 months prior, arrived at the Exercise Physiology Laboratory for a single day of data collection in which they completed health history and physical activity questionnaires, and had their blood drawn before having their body composition, cardiorespiratory fitness, and strength assessed. All data collection was completed between April 2021 and September 2021 meaning that boosters for the Moderna and Pfizer SARS-CoV-2 mRNA vaccines were not yet available for our participants.
Participants. Data were collected on 60 participants (men, n = 26; women, n = 34) of varying fitness levels including university students, faculty, staff, as well as local community members. Participant demographics are presented in Table 1. Adults between the ages of 18 and 65 who received their first and second dose of either the Moderna or Pfizer SARS-CoV-2 mRNA vaccine between 2 weeks and 6 months prior to visiting the Exercise Physiology Laboratory for data collection were eligible for the study. Participants were ineligible if they did not meet the inclusion criteria or were (a) experiencing symptoms of COVID-19 (cough, fever, shortness of breath, loss of taste or smell, chills, body aches, sore throat, diarrhea), (b) had a diagnosis of an immune-compromising condition, or (c) taking a medication that alters heart rate. Participants were apparently healthy and had no known diseases or symptoms. All research procedures were approved by the University of New Mexico Institutional Review Board prior to starting this research (IRB # 05321, approved on April 17th, 2021). This research was performed in accordance with the Declaration of Helsinki. Each participant provided written informed consent before beginning the study.
Physical activity questionnaire. The short International Physical Activity Questionnaire (IPAQ) was used to gather information about the participant's exercise and physical activity habits. The short IPAQ is a widely used questionnaire that provides a reliable and valid assessment of physical activity habits 18,19 . The questionnaire consists of seven questions related to the amount of time an individual spends exercising or working at low, moderate and vigorous levels of exertion over a typical 7-day period. When completing the questionnaire, participants were asked to recall their average 7-day level of physical activity that they had engaged in over the previous 6 months. The data gained from the questionnaire was processed accordingly to determine the average self-reported metabolic equivalent of task (MET) minutes/week 18 . Blood sample collection. Blood samples were collected through venipuncture of an arm vein into serum separator Vacutainers®. Blood samples were centrifuged at 1600xg for 15 min in 4 °C to separate serum. Samples were stored in 1 mL aliquots in a − 80 °C freezer until later analysis. Serum samples were assayed for antibodies that react with the following four SARS-CoV-2 proteins: receptor binding domain protein (RBD), spike protein subunit 1 (S1), spike protein subunit 2 (S2), and nucleocapsid (N) via MAGPIX multiplexing (Luminex xMAP Technology, San Diego, CA) and were reported according to their median fluorescence intensity (MFI). Because the SARS-CoV-2 vaccines used in this study only deliver messenger RNA that encodes for the spike protein, www.nature.com/scientificreports/ participants whose samples were positive for N protein-reactive antibodies were deemed to have been previously exposed to the live SARS-CoV-2 virus and were removed from further analyses. Intraassay coefficient of variations for S1, S2, RBD, and N antibodies were 5.35%, 4.53%, 8.95%, and 11.85%, respectively.
Anthropometric measurements. Participant height (cm) was measured using a stadiometer (Holtain Limited, Crymych, Dyfed, Great Britain) and body mass (kg) was recorded using a digital weight scale (Med-Weight MS-3900, Itin Scale Company, Brooklyn, NY, United States). Participants' body density was estimated using 3-site skinfold measurements and equations for men (chest, abdomen, and thigh 20 ) and women (triceps, suprailiac, and thigh 21 ). These measurements were used to estimate participants' percent body fat (%BF) using the Siri 22 equation.

Cardiorespiratory fitness
Maximal cardiorespiratory fitness ( V O 2max ) was estimated via the Ebbeling et al. 23 sub-maximal walking test on a motorized treadmill (Precor® C966, Woodinville, WA). Participants were fitted with a wireless heart rate monitor (Polar, Oulu, Finland) with the transmitter placed around their chest. Following a two-minute walking warm up at a slow speed (1.5 to 2 mph), the speed of the treadmill was increased to elicit a heart rate between 50 and 70% of the participants' estimated heart rate maximum (220-age). After the appropriate heart rate was reached, incline of the treadmill was increased to a 5% grade. Heart rate was recorded again at minutes 3 and 4 during this stage. If the heart rate measurements varied by more than 5 beats per minute, the test was extended for another minute and heart rate was measured again. The final two heart rates were used to calculate estimated maximal cardiorespiratory fitness according to the Ebbeling et al. 23 method.

Muscular strength.
Hand grip strength (HGS) of the dominant hand was measured using a manual springloaded dynamometer (A729-300 hydraulic hand dynamometer, Rolyan Ability One, Germantown, WI). The hand grip dynamometer was adjusted relative to the individual's hand size, so that the bar was between the proximal and middle phalanges. While standing erect, the arm and forearm were positioned such that the shoulder was adducted and neutrally rotated, elbow flexed at 90°, and forearm in a neutral position 24 . Participants were instructed to squeeze the handle of the dynamometer with maximal effort for approximately 3 s with no extraneous body movement. This process was repeated three times with 1-min rest between trials. The highest value was recorded in kilograms and normalized to body mass (kg of grip force ÷ kg of body mass) for analyses.
Using a conservative estimate of effect size (f 2 = 0.15), with one tested predictor (either %BF, relative HGS, estimated V O 2max , or MET min/week) and four covariates (age, sex, time since vaccination, and the type of mRNA vaccine received) making a total of five predictors for each model, it was estimated with an α-level of 0.05, and a power of 0.80 (1-β) that 55 participants would be required to detect a statistically significant effect of medium size on the outcome variables (S1, S2, and RBD antibodies following SARS-CoV-2 mRNA vaccination). Differences among participant demographics were evaluated using two-tailed independent sample t tests. Four separate hierarchical multiple linear regression analyses were performed to assess the main effect of an individual's %BF, relative HGS, estimated V O 2max , and self-reported MET minutes/week on the magnitude of RBD, S1, and S2 antibodies following SARS-CoV-2 mRNA vaccination. Age, sex, time since vaccination, and the type of mRNA vaccine received (Pfizer or Moderna) were included as covariates due to their known associations with prophylactic immune responses to vaccination 5,25 . These predictive models were defined as the body composition model (%BF and covariates), strength model (relative HGS and covariates), aerobic fitness model (estimated V O 2max and covariates), and the physical activity model (MET minutes/week and covariates). Dummy codes were created for sex (0 = women, 1 = men) and type of mRNA vaccine received (0 = Moderna, 1 = Pfizer). The β coefficient for each predictor variable of interest was used to quantify the magnitude of their effect. Standard error (SE) was used as a measure of the statistical accuracy of an effect size. An α of 0.05 was

Results
Model building. Case diagnostics identified two males and one female as potential influential outliers by having large Cook's distance values relative to the rest of the data set. To avoid violating the normal distribution assumption of regression analysis, these data points were excluded from further analyses. An additional seven participants were excluded from analyses as they were deemed to have likely had a recent SARS-CoV-2 infection based on high levels of antibodies against SARS-CoV-2 N (Fig. 1). Assessments of multicollinearity revealed that sex had a strong association with %BF (%BF = − 9.3 (sex) + 25.4, R 2 = 0.340, p < 0.001), relative HGS (HGS = 0.14 (sex) + 0.52, R 2 = 0.213, p < 0.001) and estimated V O 2max ( V O 2max =12.5 (sex) + 37.50, R 2 = 0.421, p < 0.001). This meant that on average, women had 9.3% more body fat, 0.14 kg less relative HGS, and an estimated V O 2max that was 12.5 ml/kg/min less than men (Table 1). To avoid violating the assumption of collinearity, sex was removed from the models that investigated the effect of %BF, relative HGS, and estimated V O 2max on the S1, S2, and RBD antibody response. No association was observed between the sex of the individual and the average amount of physical activity they engaged in each week (MET min/week = − 167.2 (sex) + 4758.7, R 2 < 0.01, p = 0.954). Of the participants in the current study, 70% (35/50) received the Pfizer SARS-CoV-2 mRNA vaccine while 30% (15/50) received the Moderna SARS-CoV-2 mRNA vaccine.

Exercise habits, physical fitness, and antibody responses.
To illustrate the range of individual's antibody responses to vaccination, Fig. 2 shows the magnitude of antibodies present in the serum following SARS-CoV-2 mRNA vaccination. To investigate the effect of modifiable physical fitness variables on these responses multiple models were developed. The body composition model (F 4,45 = 2.735, p = 0.040, R 2 = 0.195) indicated a significant effect of %BF on the amount of S1 antibody present in the serum (β = − 366.56, SE = 129.2, p = 0.006, Fig. 3A). This meant that for every 1% increase in %BF there was a significant decrease in the amount of S1 antibody regardless of the individual's age, time since vaccination, or the type of mRNA vaccine received. In the strength model (F 4,45 = 0.624, p = 0.648, R 2 = 0.052), no significant effect was observed for relative HGS on the amount of S1 antibody present in the serum (β = 1279.56, SE = 6732.23, p = 0.850). For the aerobic fitness model (F 4,45 = 1.257, p = 0.301, R 2 = 0.100), no significant effect was observed for estimated V O 2max on the amount of S1 antibody present in the serum (β = − 199.95, SE = 128.09, p = 0.125). Using the physical activity model (F 4,45 = 1.003, p = 0.427, R 2 = 0.102), no significant effect was observed for physical activity on the amount of S1 antibody present in the serum (β = 0.27, SE = 0.17, p = 0.127).
In agreement with the S1 antibody response, the body composition model (F 4,45 = 3.201, p = 0.021, R 2 = 0.221) indicated a significant effect of %BF when controlling for these covariates on the amount of RBD antibody present in the serum (β = − 249.30, SE = 80.13, p = 0.003, Fig. 3B). Using the strength model (F 4,45 = 0.681, p = 0.609, R 2 = 0.057), like the S1 antibody response, no significant effect was observed for relative HGS on the amount of RBD antibody present in the serum (β = 1612.46, SE = 4232.33, p = 0.705). Similar to the S1 antibody response, no significant effect was observed in the aerobic fitness model (F 4,45 = 0.983, p = 0.426, R 2 = 0.080) for estimated

Discussion
The primary aim of this study was to determine the effect of physical activity habits and multiple aspects of physical fitness-strength, aerobic fitness, and body composition-on the magnitude of antibody responses following SARS-CoV-2 mRNA vaccination. Poor responses to vaccination (e.g., lower viral protein specific antibody production) limits vaccine efficacy 26 and may reflect an increased risk for infectious disease morbidity and mortality 27 . The main finding of the present study was that %BF had a significant effect on the antibody response after accounting for the effects of covariates that could impact this response including age, time since vaccination, and the type of vaccine received (Fig. 3). Our statistical models demonstrated that %BF was an independent predictor of S1 and RBD antibodies present in the serum following SARS-CoV-2 mRNA vaccination. Specifically, in people with higher %BF there was a significantly lower amount of both the RBD and S1 antibodies present in the serum. While we lack sufficient evidence to suggest that an individual's %BF has a statistically significant effect on the amount of S2 antibody present in the serum (p = 0.106), we demonstrated that there was still a negative effect (β = − 190.08) of %BF on the S2 antibody response. Therefore, the effect of %BF on the S2 antibody response may indeed be of practical significance and the directionality of the relationship between %BF and S2 antibody response is in agreement with the responses observed for the S1 and RBD antibodies. The current study lends support that as %BF increases there is a decreased efficacy of SARS-CoV-2 mRNA vaccination, possibly leading to a greater risk of an individual contracting SARS-CoV-2 and developing COVID-19 regardless of their age, type of mRNA vaccine received, and days since vaccination. Therefore, although vaccination is one of our primary strategies against viruses such as SARS-CoV-2, this data suggests that higher %BF could reduce the level of circulating SARS-CoV-2-specific IgG antibodies following SARS-CoV-2 mRNA vaccination.
Obesity is characterized by chronic low-grade inflammation 28 and is a risk factor for increased morbidity and mortality amongst SARS-CoV-2 infected patients 29,30 . In one study 31 , obese high fat-fed mice developed elevated markers of inflammation that were associated with lower levels of influenza-specific neutralizing antibodies as well as defective generation of effector memory CD8 + T cells after vaccination. The authors discussed the possibility that the impaired memory T-cell response could have influenced the poorer maintenance of neutralizing antibodies observed in their study 31 . There is also evidence that obesity interferes with B-cell responses furthering the autoimmune inflammatory response following SARS-CoV-2 infection and increasing the severity of COVID-19 32 . Aside from the mentioned physiological/immunological factors that could contribute to reduced vaccine effectiveness, anatomical factors in people with higher body fatness may also reduce effectiveness. For example, as individuals increase in weight and %BF, longer needle lengths may be required in order to ensure that the vaccine is deposited intramuscularly 33 . Although vaccination amongst obese individuals will likely still result in some vaccine-conferred protection, needles that are too short and not individualized to the individual's level of body fatness may result in the vaccine being deposited into a fat pad, which could reduce the immunogenic response to the vaccine 33 . In agreement with these observations, previous researchers have demonstrated that   36,37 , and the influenza A virus [10][11][12] . Importantly, these previous vaccines elicited an immune response via inactive, attenuated, or recombinant viral proteins and are different than the two vaccines evaluated in the current study (Moderna and Pfizer) which are both lipidencapsulated mRNA vaccines. Therefore, the current study builds upon previous findings by extending them to SARS-CoV-2 mRNA vaccines (Moderna and Pfizer) and demonstrates that the effect of an individual's %BF on the magnitude of their antibody response is independent of known factors that influence vaccine-induced immunity such as the individual's age and time since vaccination 5 . Importantly, chronic low level inflammation associated with obesity impairs the generation of antibodies following influenza vaccination 31 . This may explain www.nature.com/scientificreports/ the inverse relationship between %BF and antibody responses observed in the current study following SARS-CoV-2 mRNA vaccination. Given the obesity epidemic and the fact that obesity is an independent risk factor for severe COVID-19, there is a need to enact strategies for increasing the efficacy of SARS-CoV-2 vaccines amongst this population. Based on the present results, one effective lifestyle-related strategy may be to reduce excess body fat. Although obesity can have a biological component and is therefore not always modifiable, there are behavioral and environmental factors that contribute to the development of obesity that can be modified. Proper nutritional and exercise interventions are often recommended as effective strategies for combating and preventing obesity. Decreasing excess body fat can improve the likelihood that the vaccine is deposited intramuscularly that may increase its immunogenic effect 33 . Evidence supports that decreasing excess body fat can also decrease proinflammatory markers 28 , therefore potentially protecting the generation of effector memory T-cells and neutralizing antibodies following vaccination 31,32 . Results of the current study might indicate that decreasing excess body fat can positively affect the immunogenic effect of vaccination, a notion supported by recent evidence 14 .
Participating in regular exercise and being physical active is recommended for all adults 38 . The negative health consequences of failing to meet adequate levels of physical activity are myriad and include a greater risk of COVID-19 leading to hospitalization and death 39 . Whether physical activity levels impact the efficacy of prophylactic immune response to SARS-CoV-2 vaccination is not clear, but there is growing evidence that regular physical activity increases the efficacy of vaccination 16 . While we did not observe a statistically significant effect of physical activity on the magnitude of S1 (p = 0.127), RBD (p = 0.081), and S2 (p = 0.120) antibodies following SARS-CoV-2 mRNA vaccination, in our a priori power analysis we assumed a moderate effect size (f 2 = 0.15) between our predictor and outcome variables which may have overestimated the relationship between physical activity and antibody responses resulting in the current study being slightly underpowered to identify a statistically significant effect. Though not statistically significant, there was a positive effect of the average time spent being physical active on the magnitude of antibody response for S1 (β = 0.27), RBD (β = 0.19), and S2 (β = 0.23) suggesting that physical activity may be of practical significance for improving an individual's antibody response following vaccination. In support of this, previously sedentary aged adults (~ 70 years) who were randomly assigned to a sham intervention had poorer influenza vaccine responses, while those assigned to a cardiovascular exercise program had a significantly greater seroprotection rate at 24 weeks after vaccination 40 . Also, in a recent preprint, individuals who were more physically active had a greater S1/S2 antibody response following vaccination with an inactivated SARS-CoV-2 virus vaccine (CoronaVac) 41 . The current study builds upon these findings by suggesting that increasing an individual's weekly physical activity may potentially improve the antibody response following mRNA vaccinations as well.

Conclusions
With the increasing number of SARS-CoV-2 infections and variants, along with the known waning effects of vaccination, future mRNA vaccinations such as boosters are encouraged to sustain immunity. Our findings demonstrate that as %BF increases there is a decreased efficacy of SARS-CoV-2 mRNA vaccination, characterized as decreased S1, S2, and RBD antibodies present in the serum, which can lead to a greater risk of contracting SARS-CoV-2 and developing COVID-19 regardless of the individual's age, type of mRNA vaccine received, and time since vaccination. Therefore, decreasing excess body fat may be an effective strategy to improve the prophylactic immune response following mRNA vaccination. In addition, increased physical activity may be a beneficial strategy to improve the antibody response following mRNA vaccination, but more research is warranted with larger sample sizes to substantiate this effect. These findings highlight several behavioral changes (reducing excess body fat and increasing physical activity) that individuals can take that may improve the efficacy of SARS-CoV-2 mRNA vaccination.

Data availability
The data used to support the findings of this study are available from the corresponding author upon request.